1. Field of the Invention
The invention relates to a novel treated silica which is useful for analytical sample preparation by solid phase extraction, and to a method of use of this novel silica. More specifically, the invention relates to a silica treated with phenyltrichlorosilane and with 3-mercaptopropyltrimethoxysilane, to a solid phase extraction cartridge and an extraction column comprising the treated silica of the invention, and to a method of using the silica of the invention for sample preparation and analysis. The novel silica, column and method are all particularly well suited for use in the determination of gamma hydroxybutyric acid (GHB). Gamma hydroxybutyric acid, also known as 4-hydroxybutyric acid and having the Chemical Abstracts Service Registry Number 591-81-1, is a pharmaceutical which has been used for treatment of alcohol abuse and as a treatment for narcolepsy. GHB as used herein also refers to any salt form of gamma hydroxybutyric acid. GHB is also sometimes used as a drug of abuse. GHB has also been used recently as a "date rape drug", being used to render a victim disoriented or unconscious. The disclosure of the present invention is directed to a novel method for analysis of GHB in samples, and to a novel composition useful for solid phase extraction methods used in analytical chemistry. The treated silica is believed to be functionalized with both propylsulfonic acid groups and phenyl groups as a result of the treatment.
Solid phase extraction is a method used for sample preparation in analytical chemistry. In solid phase extraction, a solid sorbent material is used to contact a solution being analyzed. An analyte of interest may be bound reversibly and selectively by the sorbent material, thereby separating it from an interfering substance which may be present in the solution. Alternatively, the sorbent material may be used to selectively bind an interfering substance present in the solution, and thereby separate that substance from the analyte of interest which is left in the solution. The treated silica of the present invention may be used as a sorbent material in either of these ways to effect separations for use in analytical chemistry.
Solid phase extraction methods are frequently carried out using a sorbent material which is carried within an extraction device such as a cartridge or a column. The extraction device is designed to be inexpensive to manufacture and therefore disposable after a single use. These extraction devices are generally tubular in shape, and sized to fit either a syringe or a vacuum manifold. The sorbent material used with such an extraction device is held in place within the cartridge or column by a frit selected for its ability to retain the sorbent material, while allowing a solvent to pass through the cartridge or column. The present invention encompasses both a cartridge extraction device and a column extraction device within its scope.
2. Description of the Related Art
Terms used in this disclosure are defined as follows:
GHB--gamma hydroxybutyric acid (also known as 4-hydroxybutyric acid ) or a salt of gamma hydroxybutyrate (also known as 4-hydroxybutyrate), wherein the salt is an alkali metal salt PA0 GBL--gamma butyrolactone PA0 GC--gas chromatography PA0 MS--mass spectrometry PA0 SPE--solid phase extraction PA0 TMCS--trimethylchlorosilane PA0 BSTFA--bis(trimethylsilyl)trifluoroacetamide--used as BSTFA/TMCS mixture PA0 TMS--trimethylsilyl PA0 D6-GHB--deuterated gamma hydroxybutyric acid PA0 SIM--selected ion monitoring PA0 C-18--octadecyl PA0 UV--ultraviolet PA0 VS--visible
GC-MS--gas chromatography-mass spectrometry PA1 HPLC--high performance liquid chromatography
At injector temperatures used for gas chromatography, GHB is readily converted to gamma butyrolactone. Lower elution temperature delays the elution of the lactone but the lactone often gives broad peaks. Procedures are known in the art for determining the lactone in urine specimens, but the lactone can be legally possessed in some jurisdictions. The free acid or salt form of GHB is a scheduled drug, that is a drug whose possession is regulated by the United States Drug Enforcement Administration. There exists an unfulfilled need for a method for determining GHB in samples such as biological samples or body fluids, which method would be definitive. That is, the method should measure GHB present in the sample which is present as either the free acid or salt, but whose results would not be affected by the presence of gamma butyrolactone in the same sample. The method should be one which can provide definitive proof of the presence of the controlled substance form of GHB in a sample. The results determined with such a method would be of great value to law enforcement officials for providing evidence of illicit drug use. Since the lactone GBL is not a controlled substance, it is highly desirable to have an analytical method for GHB which can determine GHB quantitatively without interference from GBL. In short, having GHB in a subject's urine is evidence of the commission of a crime while having GBL in the subject's urine may not be a crime. Currently, forensic and clinical laboratories analyze for GHB by extracting GHB from urine samples by conversion to GBL by acidification, followed by extraction into either chloroform or ether, and then using capillary GC analysis with either flame ionization detection or mass spectrometry to quantitate GBL. This method does not directly measure GHB however, but rather the lactone. This method then is not a definitive test for GHB, but instead measures GHB as well as any GBL in a sample. Since GBL is not a scheduled drug, this will not provide firm scientific evidence needed in court to obtain a conviction on drug charges. A definitive test for GHB, which would measure the free acid or salt in a sample, and which can distinguish GHB from the lactone GBL, would be of value to forensic laboratories.
Preferably, a method for determination of GHB should also be useful for analysis of GHB from samples other than biological samples as well. Examples of these other samples include but are not limited to oils, extracts, elixirs and beverages which might contain GHB; and which are suspected as being intended for illicit administration to people.
The short half life of GHB after administration to a subject is also a problem for detection of the drug. The half life is reported to be less than one hour. Peak urine concentrations after ingestion are observed at about 4 hours. No drug is detected after 12 hours from ingestion. Less than 5% of an oral dose is excreted in urine as the unchanged drug (S. D. Ferrara et al., Therapeutic gamma-hydroxybutyric acid monitoring in plasma and urine by gas chromatography-mass spectroscopy, Journal of Pharmacology and Biomedical Analysis, 11:6, 483-487, 1993). For an analytical method for determination of GHB to be of practical use it must be suited for rapid use when a sample suspected to contain GHB becomes available.
Currently available choices for analytical methods which might be suitable for GHB are limited. GHB has been reported to have very low absorption in the ultraviolet region. Because of this, HPLC analysis would require refractive index detection. This would clearly not provide a very sensitive detection method, and would present a serious problem with respect to obtaining sufficient resolution from interfering substances. The use of capillary electrophoresis would be faced with the same limitations of detection. Derivatization of GHB would be required to introduce a chromophore into GHB which would allow more sensitive detection using separation methods such as HPLC or capillary electrophoresis.
GHB is not readily determined by gas chromatography (GC). At temperatures above about 140.degree. C., GHB is converted to gamma butyrolactone (GBL) in an injection port. This indicates that derivatization such as silylation would be desirable to prevent formation of the lactone and adds to volatility of GHB prior to injection onto a GC column. Coupling of separation by gas chromatography with mass spectroscopic determination of the separated analytes is a problem with GHB because the mass spectra for GHB and the lactone GBL have been reported to be nearly identical. This problem has been discussed in Kathleen Andrews, Getting the Scoop on GHB: The New Recreational Drug, Workshop at 49th Annual Meeting of the American Academy of Forensic Sciences, Feb. 17-22, 1997.
The lack of suitable analytical methods for GHB which can serve as a definitive test for the presence of this drug in samples such as urine has been indicated by those working in the area (J. Letteri and H. Fung, Determination of Gamma-hydroxybutyrate acid in human urine, Journal of Pharmacology and Experimental Therapeutics, 208, 7-11, 1979).
A method for determining GHB has been reported by Andrews (Getting the Scoop on GHB: The New Recreational Drug, Workshop at 49th Annual Meeting of the American Academy of Forensic Sciences, Feb. 17-22, 1997) in which GHB is converted to its lactone prior to extraction by heating under acidic conditions. This method used an internal standard, .alpha.-methylene-.gamma.-butyrolactone. The extracts were subjected to capillary gas chromatography for quantitation of the GBL present. This method suffers the disadvantage of not being definitive for GHB. That is, this method does not measure GHB itself in a sample but rather measures GHB in addition to any GBL which may be present in the sample. This reported method comprises the steps: a) adding the internal standard to a sample; b) adding a quantity of sulfuric acid for acidification; c) centrifuging the sample; d) decanting the supernatant; e) adjusting the pH to between 6 and 7; f) centrifuging again; g) adding n-butyl chloride to the supernatant; h) centrifuging again; i) removing the solvent layer; j) reducing the solvent volume by evaporation; and k) submitting a sample of the solvent layer to gas chromatography on a capillary column with flame ionization detection.
As disclosed in M. Z. Mesmer and R. D. Satzger, Determination of Gamma-Hydroxybutyrate (GHB) and Gamma-Butyrolactone (GBL) by HPLC/UV-VIS Spectrophotometry and HPLC/Thermospray Mass Spectrometry, in J. of Forensic Sci., 43(3), 489-492, May, 1998, GHB may be determined by two HPLC methods. The first of these methods consists of HPLC on a C-18 reversed phase column with UV (ultraviolet) detection at 215 nm. This method would be applicable only with samples which had a very low content of UV absorbing substances. The second method disclosed in Mesmer consists of HPLC separation coupled with mass spectrometry. This method disclosed by Mesmer was indicated in the disclosure as suited for application to "illegal preparations that are available on the black market" which might contain GHB. This second method provided in this disclosure has a sample of a suspected illegal preparation subjected to separation by HPLC on a C-18 reversed phase column, coupled with thermospray mass spectrometry for detection of GBL and GHB resolved by the column. The method disclosed was described by the authors as "capable of differentiating between GHB and its lactone, GBL", but was described as having sensitivity for analysis of illegal preparations only. The method disclosed by Mesmer was not indicated as having enough sensitivity to allow detection in samples taken from a human subject suspected as having ingested GHB. A sample of GHB suitable for illicit sale would be expected to have a high concentration of GHB, although some inactive diluent might also be present. The concentration in an illegal preparation for sale would certainly be much higher than would be expected for a sample taken from a subject suspected to have ingested GHB. This disadvantageous lack of sensitivity is believed to be overcome by the method of the present invention and the use of the novel silica disclosed herein.
As disclosed in K. M. Gibson et al., Stable Isotope Dilution Analysis of 4-Hydroxybutyric Acid: An Accurate Method for Quantification in Physiological Fluids and the Prenatal Diagnosis of 4-Hydroxybutyric Aciduria, in Biomed. and Environ. Mass Spectrometry, 19, 89-93, 1990; GHB may be determined quantitatively by use of isotope dilution with deuterated GHB being used. In this publication the analyte being determined was the butyrolactone, GBL. That is, GHB in a sample was converted in the method of Gibson to GBL by acidification. GBL was then separated on a silicic acid column from other substances present in the sample. And subsequently, GBL eluted from the silicic acid column was trimethylsilylated for further separation and quantitation by GC-MS. Deuterated GBL (D6-butyrolactone) was added to the sample before acidification and adsorption with silicic acid to provide for isotope dilution. This method suffers from the disadvantage that GHB is not directly determined. That is, the method of Gibson is not definitive for GHB. According to the method disclosed in Gibson, a sample is acidified for conversion of any GHB in the sample to GBL. This step allows the use of D6-GBL as a stable isotope dilution internal standard to allow for any sample loss which might occur during the steps of the method. The method of Gibson then performs a separation of GBL by chromatography on silicic acid. By this method then, a sample which might originally contain the lactone, GBL, but no GHB; would give a result similar to that which would be obtained for a sample which would originally contain an ecluivalent amount of GHB and no lactone. Therefore, the method disclosed by Gibson is not definitive for GHB, and might not provide suitable scientific evidence for supporting criminal charges of illicit use of GHB.
All of the reported methods for analysis of GHB therefore have limitations or shortcomings to their use for forensic use.